The present invention relates to immunological materials and methods useful for the determination of dihydropyrimidine dehydrogenase (DPD) in biological samples.
Dihydropyrimidine dehydrogenase (DPD) is an enzyme which catalyzes the reduction of pyrimidines to 5,6-dihydropyrimidine, where the resulting catabolites, dihydrouracil and dihydrothymine, are further catabolized to .beta.-alanine or .beta.-aminoisobutylic acid, respectively.
DPD is also known to catalyze the reducing reactions of 5-fluorouracil (5-FU) and various catabolites from pyrimidine analogues and derivatives, which are widely utilized as antitumor medicines, such as 2'-deoxy-5-fluorouridine (2'-dFUrd), 5'-deoxy-5-fluorouridine (5'-dFUrd, doxifluridine), and the like. It has been reported that the DPD level in biological samples of individuals suffering from tumor is an interesting marker of the therapeutic efficacy of antitumor medicaments in the series of 5-fluorouracil derivatives (see e.g. Iigo et al., 1969, Biochem. Pharmacol., 38, 1885-1889).
Deficiency in DPD is known to be responsible for the toxicity of the metabolites of pyrimidine antitumor medicines, which results in life-threatening condition during chemotherapy (Milano, G. and Etienne, 1988, M.C., Pharmacogenetics, 4, 301-306). Recently it was found that the disorder of DPD deficiency is more frequent than initially thought (Diasio and Lu, 1994, J. Clin. Oncol., 12, 2239-2242). It is thus important to identify patients with DPD deficiency.
There is therefore a need for a sensitive and reliable assay for the determination of the DPD level in biological samples.
Several methodologies for such assays have been described. Femandez-Salguero et al. reported a thin layer chromatography (TLC) procedure for the determination of DPD activity in human peripheral lymphocytes (Femandez-Salguero et al., 1995, Biochem. Pharm. 50, 1015-1020). The assay uses radiolabeled uracil as a substrate. However, a method which uses a radiolabel is obviously not adapted to routine diagnosis in hospitals. Furthermore, such a TLC method is time-consuming.
Other assay methods utilizing HPLC have been also described, e.g. by van Gennip et al., 1982, Adv. Exp. Med. Biol., 253A: 111-118, and Sommadossi et al., 1982, J. Biol. Chem., 257: 8171-8176. Those methods are based on the measurement of enzyme activity by the determination of the sum of various catabolites of 5-FU using reverse phase HPLC. Such methods are cumbersome and time-consuming.
The problem addressed by the present invention is to find materials and methods for the determination of DPD level that do not have the drawbacks of the above known methods, and that provide a simple and speedy assay for routine use in hospitals.